Gene therapy is the process of inserting genes into a person's cells to treat disease. Recent advances have led to successful clinical trials for diseases like retinal disorders, cancers, and genetic disorders. There are two main strategies for gene therapy - gene augmentation replaces a missing gene, while gene inhibition therapy blocks a dominant gene. Viruses are commonly used as vectors to deliver therapeutic genes. While promising, gene therapy still faces challenges like short-lived effects, immune responses, and difficulties treating multi-gene disorders and cancers located in multiple sites.

1. GENE THERAPY
Presented by:
Jacob Thon Bior
M. Pharm 1st year
Dept of Pharmaceutical Chemistry.
KLE College of Pharmacy, Belagavi.
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2. Content:
Introduction.
Advances in gene therapy.
Principle of DNA Estimation.
 Principle of RNA Estimation.
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3. Gene Therapy
Is the process of inserting gene into person’s cells to treat or prevent diseases. It is novel approach
to treat, cure and prevent by changing the expression of person’s genes. In future, it will help
doctor to treat by inserting genes instead of drugs or surgery.
Dreams comes true for researchers to replace mutated gene that causes diseases with healthy copy
of genes and cure the genetic disorders.
Due to advance in biochemistry and molecular biology which help in understanding genetic
basis of inherited diseases although the early clinical failure almost dismiss gene therapy the 2006
success has given a promise. This include successful treatment of people with retinal disease, X-
linked SCID, Adrenoleukodystropy, chronic lymphocytic leukemia, acute lymphocytic
leukemia, multiple myeloma and Parkinson's disease.
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4. Strategies
Gene Augmentation therapy:
DNA is inserted to the genome to replace missing gene product.
Gene Inhibition Therapy:
The antisense gene inhibits the expression of dominant gene.
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5. Types of Gene Therapy
 Somatic gene therapy :
The therapeutic genes are transferred into the somatic cells, or body, of a
patient. Any modifications and effects will be restricted to the individual patient
only, and will not be inherited by the patient's offspring or later generations.
Germ line gene therapy :
Sperm or eggs, are modified by the introduction of functional genes, which are
integrated into their genomes. This would allow the therapy to be heritable and
passed on to later generations.
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6. Recent Advancing on Gene Therapy
It depends on delivery of correct genes, viral vectors are tools use by molecular
biologist to deliver genetic material into the cell. This process can be performed
inside living organism (in vivo) or in cell culture(in vitro). Viruses have evolved
specialized molecular mechanism to effective transport their genomes inside the cell
they infected.
Viruses use as vector to introduce the genetic material inside the bodies, this viruses
are inactivated hence do not reproduce. E.g. Adenoviruses, Adeno-associated virus,
Herpes virus (DNA tumor virus), Retrovirus (RNA tumor virus), Lenti virus, pox
virus and measles virus.
1. Non viral vector:
This include the use of liposomes (less immunogenic) and use of nanotechnology(
nanometer size)
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2.‘Mending broken hearts' by using gene therapy Novel techniques to “mend broken
hearts” using gene therapy and stem cells represent a major new frontier in the treatment of heart
disease. It was achieved by the researchers at Gladstone Institute of Cardiovascular Disease in
California, They were able to re-programmed scar-forming cells into heart muscle cells, some of
which were capable of transmitting the kind of electrical signals that make the heart beat, They
performed on a live mice, transforming scar-forming cells, called fibroblasts, into beating heart
muscle cells. They injected three genes (cocktail of genes) into the heart of live mice that had
been damaged by heart attack, fibroblasts could be turned into working heart cells. Researchers
said that the “cocktail of genes” used to regenerate cells could one day be replaced with “small
drug-like molecules” that would offer safer and easier delivery
3.Mucopolysaccharidosis Type IIIA potential gene therapy. Mucopolysaccharidosis
Type IIIA (MPSIIIA) is a metabolic disorder in which the body is missing an enzyme required to
break down long chains of sugars known as glycosaminoglycans. The glycosaminoglycans
accumulate in body and cause damage, particularly in the brain if not broken. Fatima Bosch at
university of Barcelona in Spain developed a form of gene therapy to replace the missing enzyme
in MPSIIIA,They injected the replacement gene into the cerebrospinal fluid that surrounds the
brain and spinal cord. This study demonstrates that gene therapy can be delivered to the brain
through the cerebrospinal fluid.

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4. Stem cell gene therapy gives hope to prevent inherited neurological disease.
Scientists from The University of Manchester have used stem cell gene therapy to
treat a fatal genetic brain disease.
It was used to treat Sanfilippo – a fatal inherited condition which causes progressive
dementia in children.
Sanfilippo, is currently untreatable mucopolysaccharide (MPS) disease
It is caused by the lack of SGSH(N-sulfoglucosamine sulfohydrolase) enzyme in
the body which helps to breakdown and recycle long chain sugars, such as heparan
sulphate (HS).
Children with the condition build up and store excess heparan sulphate (HS)
throughout their body from birth which affects their brain and results in progressive
dementia and hyperactivity, followed by losing the ability to walk and swallow.

9. 5. UCLA researchers combine cellular and gene therapies to develop treatment
for breast cancer
Carol Kruse, a professor of neurosurgery and member of the Jonsson Cancer
Center and the UCLA Brain Research Institute led the research on breast cancer.
Breast cancer is the most common form of cancer in women, and metastasis is a
major cause of health deterioration and death from the disease.
Cellular therapy and gene therapy were used together to treat breast cancer. Cellular
therapy is a type of immunotherapy that uses T cells, the foot soldiers of the
immune system, that have been sensitized in the laboratory to kill breast cancer
cells.
These sensitized T cells are injected into the parts of the brain to which cancer has
spread.
The research shows that the T cells can move through tissue and recognize and
directly kill the tumor cells.
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6.First Real-Time MRI-Guided Gene Therapy for Brain Cancer.
Neurosurgeons at the University of California, San Diego School of Medicine and UC San
Diego Moores Cancer Center are among the first in the world to utilize real-time magnetic
resonance imaging (MRI) guidance for delivery of gene therapy as a potential treatment for brain
tumors.
Using MRI navigational technology, neurosurgeons can inject Toca 511 (vocimagene
amiretrorepvec), a novel investigational gene therapy, directly into a brain malignancy.
The new approach offers a precise way to deliver a therapeutic virus designed to make the tumor
susceptible to cancer-killing drugs.
Toca 511 is a retrovirus engineered to selectively replicate in cancer cells, such as glioblastomas.
Toca 511 produces an enzyme that converts an anti-fungal drug, flucytosine (5-FC), into the anti-
cancer drug 5-fluorouracil (5-FU).
Cancer cell killing takes place when 5-FC comes into contact with cells infected with Toca 511.

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IS GENE THERAPY TOTALLY SAFE ??
Although gene therapy is a promising treatment option for a number of diseases (including
inherited disorders, some types of cancer, and certain viral infections), the technique remains risky
and is still under study to make sure that it will be safe and effective.
Gene therapy is currently only being tested for the treatment of diseases that have no other cures.
Technical Difficulties in Gene Therapy
Gene delivery: Successful gene delivery is not easy or predictable, even in single-gene disorders.
For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus
in the lungs makes it physically difficult to deliver genes to the target lung cells.
Delivery of genes for cancer therapy may also be complicated by the disease being present at
several sites.
Gene-therapy trials for X-linked severe combined immunodeficiency (X-SCID), however, have
been more successful.

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Problems with Gene Therapy
Short Lived:
Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells
prevent gene therapy from long time Would have to have multiple rounds of therapy
Immune Response:
An organism introduced leads to immune response increased response when a repeat offender
enters the gene might be over-expressed (toxicity)
Viral Vectors:
Patient could have toxic, immune, inflammatory response also may cause disease once inside
Multigene Disorders:
Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because
you need to introduce more than one gene

13. DNA
Principle :
This reaction is generally given by deoxypentose, the 2-deoxyribose of DNA, in
present of acid is converted to β-hydroxilevulinic aldehyde which then react with
diphenylamine to form blue colored complex which is read at 595nm
The intensity of blue color is directly proportional to concentration of DNA.
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14. Procedure:
Dissolve 1.5g diphenylamine in 100ml of glacial acetic acid, add 1.5ml of
concentrated sulphuric acid store the solution in dark glass bottle. On the day of use
prepare a fresh solution of ethanal (1ml) in 50ml water. Add 0.5ml of this solution
to each 100ml of diphenylamine solution.
ASSAY:
•Prepare series of dilutions of standard DNA (0.25mg/ml) in saline citrate buffer to
give concentration of 50-500μg/ml.
•Prepare all samples in triplicate.
•To 2ml of each dilution of blank, standard and unknown add 4ml of diphenylamine
reagent and mix.
•Warm, cool and read absorbance at 595nm against the blank.
•Construct the standard curve and calculate the concentration of unknown DNA
sample dissolve in saline citrate solution.
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15. RNA
Principle:
This is a general reaction of pentose and depends on the formation of furfural when pentose is
heated with concentrated hydrochloric acid, orcinol react with furfural in present of ferric chloride
as catalyst to give green color which is measured at 665nm.
The absorbance is directly proportional to concentration of RNA.
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16. Procedure:
•Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1ml of working standard into series of labeled
test tubes. Pipette out 1ml of given sample into other test tubes. Makes up the
volume to 1ml in all test tubes, test tube with 1ml act as blank. Add 2ml of orcinol
reagent to all test tube including blank. Mix by shaking and heat on boiling water
for 20 minutes. Cool the content and record the absorbance at 665nm against blank.
Plot standard curve by taking concentration of RNA on X-axis and absorbance at
665nm on Y-axis. From this standard curve calculate the concentration of RNA in
given sample ( μg RNA /ml).
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17. REFERENCE:
Biochemistry by U. Sathyanarayan
Page:625-630
Introductory practical biochemistry, edited by S.K. Sawhney and
Randhir Singh , Narosa pub-house page: 74-75
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